1. Field of the Invention
The present invention relates to a microscopy method and a microscope, which use electromagnetic rays of two wavelengths. This application is based on Japanese Patent Application No. 2006-349694, the content of which is incorporated herein by reference.
2. Description of the Related Art
The technology of the optical microscope has a long history, and various types of microscopes have been developed. Moreover, in recent years, a further highly functional microscope system is being developed due to advancement of peripheral technologies including laser and electronic imaging technologies. Particularly, in the field of nanobioscience, there has been developed a microscopic measurement method which uses illuminating lights of two different wavelengths. In this microscopic measurement method, two laser beams of different wavelengths are overlapped and converged onto a specimen simultaneously, and photoresponsive data such as scattering light and fluorescence from the specimen is measured. The method attracts attention as a measurement method to analyze optical responses in the time domain as well as spatial measurement simultaneously.
A known typical microscopic measurement method uses plural light sources to irradiate pulsed laser beams of different wavelength onto a specimen, and then detects photoresponsive signals including fluorescence emitted from the irradiated domain. One example of such a method is an IR-visible double resonance microscopy using double resonance of infrared light and visible light (For example, refer to Japanese Patent Number 3020453).
In this IR-visible double resonance microscopy, fluorescent molecules in the ground state S0 is excited to high-vibrational excited state S0′ belonging to the ground state by irradiation of infrared light as shown in FIGS. 9 (a) and (b). The activated molecules are further activated to upper electron-excited state S1 by irradiating visible light. The molecules thus activated to S1 state generate fluorescence, and relax back to the ground state S0.
In this IR-visible double resonance microscopy, for example, pulsed infrared light and pulsed visible light are focused at the same region of a specimen, and the focus points are two-dimensionally scanned on the specimen relatively and fluorescence signals at each focus point are measured to form two-dimensional fluorescence image in a computer. In this case, fluorescence cannot be produced unless S0′ state of the molecules is generated by the resonance with the infrared light, therefore the obtained fluorescence image shows spatial distribution of the molecules in infrared vibrationally excited state.
The characteristic of this microscopy method is that it can observe spatial distribution of the molecules in the infrared vibrationally excited state with the resolution of visible light (i.e. a few to several hundreds of nano meters), while the spatial resolution in conventional infrared microscopic spectroscopy is limited by infrared diffraction limit, i.e. a few to several microns.
An example to detect distribution of CH groups in rhodamine 6G molecules included in a specimen will be discussed below, as a specific example. A rhodamine 6G molecule has side-chain CH groups as shown in the molecular structure in FIG. 10(a), and it has an absorption band, for example, in the vicinity of 3200 cm−1 (wave length: 3.1 μm, photon energy: 0.4 eV) due to CH breathing vibration. In addition, the wavelength corresponding to the transition/absorption from the ground state S0 to electron-excited state S1 is 532 nm (photon energy: 2.33 eV).
Thus, rhodamine 6G molecules are excited to the vibration-rotation level S0′, where v=1, belonging to the ground state by means of infrared ray with wavelength of 3.1 μm as shown in the diagram in FIG. 10 (b). Then, they are doubly-excited by a laser beam (visible light) with the wavelength (approx. 640 nm) corresponding to the energy gap from the S0′ state to the S1 state, i.e. 1.9 eV.
As a result, rhodamine 6G molecules finally reach the electron-excited state S1, and then they will emit fluorescence and relax to the ground state S0. This fluorescence process does not occur unless the visible light and the infrared light overlap each other on the specimen in terms of space and time. In addition, both wavelengths of the visible light and the infrared light need to correspond to the energy gaps or wavelength between molecular quantum states. In other words, the visible light and the infrared light must satisfy the condition of double resonance absorption of the rhodamine 6G molecules.
Thus, the fluorescence is not detected when the infrared light does not exist. Since infrared absorption by the CH breathing vibration occurs and the fluorescence is emitted only when both the infrared light and the visible light exist, the fluorescence image to be obtained is equivalent to the visualized spatial distribution of vibrational excited state of the CH groups.
In general, molecules have various chemical groups besides CH group such as OH, SH, NH and the like, and each of these chemical groups has its characteristic resonant frequency. Therefore, if wavelengths of the visible light and the infrared light are synchronized for each chemical group, it will be possible to obtain a fluorescence image corresponding to spatial distribution of the each chemical group.
The spatial region from which the fluorescence signals are emitted is an overlap region of the visible and infrared lights despite the optical response of the specimen being in the infrared domain, therefore the spatial resolution of the fluorescence image to be obtained is determined by the diffraction limit of the visible light. For example, if the wavelength of a visible light is 500 nm and a numerical aperture of an object lens of a microscope is 1.4, spatial resolution of nearly 200 nm can be achieved.
Even more particularly, in terms of spatial resolution, it is also possible to configure the microscope to have depth resolution. More specifically, because the fluorescence signals are obtained only from the vicinity of the focal plane where visible light and infrared light are converged simultaneously with sufficient intensity, three-dimensional cross-sectional image can also be obtained by moving the specimen along optical axis with respect to the focus position.
In addition, if the light sources of the visible light and the infrared light are pulsed light sources, fluorescence signals are obtained only when these light sources are overlapped also in a time domain. Therefore, by shifting the timing of pulsed oscillations between the visible light and the infrared light, it will also be possible to trace a time response in regard to a relaxation process of a vibrational-excited state.
However, according to the experimental examination conducted by the present inventor, it becomes clear that there are some points to be improved in detection methods of the conventional light response signal, as described below. At first, because the detection methods described above basically detect the fluorescence, the molecules as observation objects are required to have high fluorescence efficiency. Consequently, in a living specimen, observation objects are limited to autofluorescent molecules. Moreover, in order to observe non-autofluorescent molecules, the observation object need to be, for example, stained by a fluorescent dye.
In addition, because molecules excited to S1 state can emit fluorescence of one photon per one molecule theoretically, the quantity of detection signals is determined by the number of excitation and fluorescence yield during the light irradiation. Moreover, during the excitation cycle, discoloration of molecules occurs and therefore the quantity of signals is suppressed. As a result, the S/N ratio may deteriorate remarkably, and in order to improve the S/N ratio, longer measurement time and/or higher light source intensity will be required.